We focus on the first step of miRNA biogenesis, where most of the regulations take place. Drosha initiates miRNA biogenesis by chopping hairpin-shaped miRNA precursors (pre-miRNA) off the primary transcripts (pri-miRNA) in the nucleus. Reduced enzymatic activity of Drosha has been described in various malignancies. We have created a powerful genome-wide genetic screen approach based on CRISPR. Applying this method in addition to data-analysis of the TCGA database, we plan to identify novel cellular regulators of Drosha activity and investigate why and how these are regulated in physiology and in cancer. Their impacts on the efficiency and accuracy of miRNA biogenesis will then be tested by in the corresponding KO cells. DNA-directed RNAi (shRNA expressed from plasmids) is more desirable than traditional synthetic siRNAs in setting of gene therapy. However, unsatisfactory knockdown efficacy and off-target effects hamper its applications, often due to inefficient, low fidelity of processing. Our previous finding on Dicer processing has established a loop-counting rule, which laid the groundwork in designing Pol III-driven pre-miRNA-like shRNAs free of the off-target effects resulting from heterogeneous processing. We are currently working on transferring such a design into the Pol II system, in which more complicated manipulation of shRNA function is possible. In particular, we are developing conditionally activated shRNAs whose function can be specifically turned on in cancer cells. This approach will dramatically increase shRNA specificity and safety. Given the long half-lives of mature miRNAs (ranging from hours to days), biogenesis control by itself is inadequate in situations that require rapid changes in miRNA function. Post-maturation regulation is an important component of how miRNAs function. Thus, in alignment with our goal of understanding miRNA regulation, we study the biogenesis and function of 3' isomiRs, which are miRNA variants generated by post-maturation tailing (adding nucleotides) and/or trimming (removing nucleotides). Interestingly, the alteration in isomiR profile rather than in overall miRNA abundance correlates with cancer progression, which suggests a unique role of isomiRs in tumorigenesis. These observations highlight the importance of isomiR study. To investigate the function of 3' isomiR, we checked the status of 3' sequence modifications during miRNA overexpression and decay. We found that isomiR profiles are not random but rather tightly regulated. This suggests that the 3' end modification is not merely a consequence of miRNA overexpression, but rather plays an active role in maintaining the miRNA hemostasis. To establish a causative relation, we are working on identifying the enzymes that are responsible for producing certain isomeric forms. We also aim to develop novel strategies to monitor the function specific to certain isomiRs. Overall, these studies seek to significantly advance our basic understanding of isomiRs, and provide a foundation for future mechanistic study of their functions in cancer.